Zahoránszky LA, Katona GY, Hári P, Málnási-Csizmadia A, Zweig KA, Zahoránszky-Köhalmi G.

Breaking the hierarchy--a new cluster selection mechanism for hierarchical clustering methods.

Algorithms Mol Biol. 2009 Oct 19;4:12. doi: 10.1186/1748-7188-4-12.

Felföldi G, Marokházi J, Képiró, M., Venekei, I.

Identification of natural target proteins indicates functions of a serralysin-type metalloprotease, PrtA, in anti-immune mechanisms

Appl Environ Microbiol. 2009 May;75(10):3120-6. Epub 2009 Mar 20.

Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involving Manduca sexta and Photorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: beta-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of serralysins of vertebrate pathogens might be found among the components of the innate immune system.

Sarlós, K., Gyimesi, M., Kovács, M.

Anyagmozgatás és információ-továbbítás a sejtben: biológiai motorok sokfélesége.

Természet Világa, 2009 május

Takács, B., Kovács, M.

Motorok a sejtben - Mi hajt bennünket?

Élet és Tudomány, LXIV/6.

Zeke A, Lukács M, Lim WA, Reményi A.

Scaffolds: interaction platforms for cellular signalling circuits.

Scaffold proteins influence cellular signalling by binding to multiple signalling enzymes, receptors or ion channels. Although normally devoid of catalytic activity, they have a big impact on controlling the flow of signalling information. By assembling signalling proteins into complexes, they play the part of signal processing hubs. As we learn more about the way signalling components are linked into natural signalling circuits, researchers are becoming interested in building non-natural signalling pathways to test our knowledge and/or to intentionally reprogram cellular behaviour. In this review, we discuss the role of scaffold proteins as efficient tools for assembling intracellular signalling complexes, both natural and artificial.

Hajdú, I., Szilágyi, A., Kardos, J., and Závodszky, P.

A link between hinge-bending domain motions and the temperature dependence of catalysis in IPMDH

Biophys J. 2009 Jun 17;96(12):5003-12.

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k(cat)/K(M,IPM)) for the substrate IPM has an unusual temperature dependence, showing a local minimum at approximately 35 degrees C. In search of an explanation, we measured the individual constants k(cat) and K(M,IPM) as a function of temperature, and found that the van 't Hoff plot of K(M,IPM) shows sigmoid-like transition in the 20-40 degrees C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30 degrees C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

Forgacs E, Sakamoto T, Cartwright S, Belknap B, Kovács, M., Tóth J, Webb MR, Sellers JR, White HD.

Switch 1 mutation S217A converts myosin V into a low duty ratio motor.

J Biol Chem. 284(4):2138-49.

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-Pi (inorganic phosphate) and actomyosin V-ADP-Pi and the rate of the hydrolysis step (myosin V-ATP-->myosin V-ADP-Pi) were all approximately 10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from approximately 0.85 in WT myosin V to approximately 0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.

Süveges, D., Gáspári, Z., Tóth, G., Nyitray, L.

Charged single alpha-helix: a versatile protein structural motif.

Proteins. 74(4):905-16.

A few highly charged natural peptide sequences were recently suggested to form stable alpha-helical structures in water. In this article we show that these sequences represent a novel structural motif called "charged single alpha-helix" (CSAH). To obtain reliable candidate CSAH motifs, we developed two conceptually different computational methods capable of scanning large databases: SCAN4CSAH is based on sequence features characteristic for salt bridge stabilized single alpha-helices, whereas FT_CHARGE applies Fourier transformation to charges along sequences. Using the consensus of the two approaches, a remarkable number of proteins were found to contain putative CSAH domains. Recombinant fragments (50-60 residues) corresponding to selected hits obtained by both methods (myosin 6, Golgi resident protein GCP60, and M4K4 protein kinase) were produced and shown by circular dichroism spectroscopy to adopt largely alpha-helical structure in water. CSAH segments differ substantially both from coiled-coil and intrinsically disordered proteins, despite the fact that current prediction methods recognize them as either or both. Analysis of the proteins containing CSAH motif revealed possible functional roles of the corresponding segments. The suggested main functional features include the formation of relatively rigid spacer/connector segments between functional domains as in caldesmon, extension of the lever arm in myosin motors and mediation of transient interactions by promoting dimerization in a range of proteins.

Pál-Gábor, H., Gombos, L., Micsonai, A., Kovács, E., Petrik, É., Kovács, J., Gráf, L., Fidy, J., Naiki, H., Goto, Y., Liliom, K., Kardos, J.

Mechanism of lysophosphatidic acid-induced amyloid fibril formation of β2-microglobulin in vitro under physiological conditions

Biochemistry. 48(24):5689-99

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Good, M., Tang, G., Singleton, J., Reményi, A., Lim, W.A.

The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.

Cell. 136(6):1085-97.

The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3s activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7.